Summary ? Project 2 Our working hypothesis, which is based on our publications and our preliminary data, is that the median preoptic nucleus (MnPO) contributes to CIH hypertension due to activity dependent changes in gene expression mediated by FosB that are driven by circulating angiotensin II (ANG II) working through the subfornical organ (SFO). The MnPO influences sympathetic nerve activity through its projection to the paraventricular nucleus of the hypothalamus. We have identified several putative FosB target genes in MnPO that may contribute to CIH hypertension including angiotensin converting enzyme 1 (ACE1), and based on our preliminary data the angiotensin receptor type 1a (AT1aR). Together these FosB target genes may drive the MnPO contribution to CIH hypertension through their possible interaction with the sodium-potassium-chloride co-transporter 1 (NKCC1). Specific Aim 1: to determine the contribution of ACE1 in the MnPO to CIH mediated hypertension. Hypothesis: Increased expression of ACE1 in the MnPO is necessary for CIH hypertension Specific Aim 2: to test the role of increased AT1aR expression in the MnPO in CIH hypertension. Hypothesis: Increased AT1aR expression contributes to enhanced activation of the MnPO during CIH. Specific Aim 3: to determine the role of NKCC1 in the MnPO in CIH hypertension. Hypothesis: Increased expression and activity of NKCC1 reduces the inhibitory effects of GABA in the MnPO leading to greater sympathoexcitation during CIH. These hypotheses will be tested using an integrative approach combining whole animal experiments employing virally mediated shRNA knockdown, in vivo sympathetic nerve and single unit recording, in vitro patch clamp recording, and laser capture microdissection with qRT-PCR. Optogenetic approaches will be used to test the role of the SFO-MnPO pathway in CIH hypertension using promoters that are specific for glutamate expressing neurons.